Publications by authors named "A Cort��s"

2 Publications

  • Page 1 of 1

Efficient and Precise Genome Editing in with Recombineering and CRISPR/Cas9-Mediated Counter-Selection.

ACS Synth Biol 2019 08 18;8(8):1877-1889. Epub 2019 Jul 18.

BioTechnology Institute and Department of Plant and Microbial Biology , University of Minnesota-Twin Cities , St. Paul , Minnesota 55108 , United States.

Dissimilatory metal-reducing bacteria, particularly those from the genus , are of importance for bioremediation of metal contaminated sites and sustainable energy production. However, studies on this species have suffered from a lack of effective genetic tools for precise and high throughput genome manipulation. Here we report the development of a highly efficient system based on single-stranded DNA oligonucleotide recombineering coupled with CRISPR/Cas9-mediated counter-selection. Our system uses two plasmids: a sgRNA targeting vector and an editing vector, the latter harboring both Cas9 and the phage recombinase W3 Beta. Following the experimental analysis of Cas9 activity, we demonstrate the ability of this system to efficiently and precisely engineer different strains with an average efficiency of >90% among total transformed cells, compared to ≃5% by recombineering alone, and regardless of the gene modified. We also show that different genetic changes can be introduced: mismatches, deletions, and small insertions. Surprisingly, we found that use of CRISPR/Cas9 alone allows selection of recombinase-independent mutations, albeit at lower efficiency and frequency. With synthesized single-stranded DNA as substrates for homologous recombination and Cas9 as a counter-selectable marker, this new system provides a rapid, scalable, versatile, and scarless tool that will accelerate progress in genomic engineering.
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August 2019

A new recombineering system for precise genome-editing in Shewanella oneidensis strain MR-1 using single-stranded oligonucleotides.

Sci Rep 2019 01 10;9(1):39. Epub 2019 Jan 10.

BioTechnology Institute and Department of Plant and Microbial Biology, University of Minnesota-Twin Cities, St. Paul, MN, 55108, USA.

Shewanella oneidensis MR-1 is an invaluable host for the discovery and engineering of pathways important for bioremediation of toxic and radioactive metals and understanding extracellular electron transfer. However, genetic manipulation is challenging due to the lack of genetic tools. Previously, the only reliable method used for introducing DNA into Shewanella spp. at high efficiency was bacterial conjugation, enabling transposon mutagenesis and targeted knockouts using suicide vectors for gene disruptions. Here, we describe development of a robust and simple electroporation method in S. oneidensis that allows an efficiency of ~4.0 x 10 transformants/µg DNA. High transformation efficiency is maintained when cells are frozen for long term storage. In addition, we report a new prophage-mediated genome engineering (recombineering) system using a λ Red Beta homolog from Shewanella sp. W3-18-1. By targeting two different chromosomal alleles, we demonstrate its application for precise genome editing using single strand DNA oligonucleotides and show that an efficiency of ~5% recombinants among total cells can be obtained. This is the first effective and simple strategy for recombination with markerless mutations in S. oneidensis. Continued development of this recombinant technology will advance high-throughput and genome modification efforts to engineer and investigate S. oneidensis and other environmental bacteria.
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January 2019